The present invention relates to improved methods of quantifying nucleic acids that are unique to a transgenic corn event designated Bt11 in a biological sample and to compositions useful in performing the methods.
Consequential to the implementation of regulations surrounding transgenic crop plants, for example European Commission (EC) Regulation 1139/98, EC Regulation 49/2000, and EC Regulation 50/2000, there has been the need to measure accurately the levels of DNA from a transgenic species that may be present: in for example, grain used for food. Analytical methods to detect and quantify DNA from these transgenic plants have received much attention particularly because the threshold value for labeling, set down by the European Commission's Standing Committee in 1999, is 1%.
It is possible to detect the presence of a transgene by any well-known nucleic acid detection method including but not limited to thermal amplification (polymerase chain reaction (PCR)) using polynucleotide primers or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA construct that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, for example, promoters, terminators, and marker genes, because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods may not be useful for discriminating between different transgenic events, particularly those produced using the same DNA construct.
To distinguish between transgenic events, event-specific PCR methods have been developed, insertion of a heterologous DNA construct into a plant's genome creates unique event-specific junctions between the integrated DNA sequence and the plant's genomic sequence. Event-specific quantitative PCR (qPCR) methods have been developed for transgenic events, including one for Bt11 Factors that may limit the applicability for such methods may include, for example, influences of initial DNA concentration, standards set by regulatory agencies, selection of primers and PCR protocol, repeatability from sample to sample, reproducibility between different laboratories, and thresholds for low level detection and high sensitivity.
For the foregoing reasons, there is a need for improving the quantitative detection of nucleic acids from the Bt11 transgenic corn event in a biological sample.